Expression of reverse transcriptase genes in Fulvia fulva.

Antibodies raised against intercellular fluid antigens isolated from diseased tomato leaves have revealed that the fungal pathogen Fulvia fulva expresses genes for a fungal reverse transcriptase (RNA-dependent DNA polymerase). This enzyme is required for the replication of retroviruses and retroviral-like transposable elements and could provide a mechanism for increasing the mutation rate of fungal pathogens, perhaps explaining their ability to evolve new races rapidly. We report here the DNA sequence of a 225-bp clone from a lambda gt11 genomic library of F. fulva. This clone, designated P5, exhibits a high degree of sequence homology with the reverse transcriptase (pol) gene of the Drosophila melanogaster copia-like retrotransposon 17.6. Southern blot analysis of genomic DNA of F. fulva showed that P5-related sequences are moderately reiterated with 30-100 copies, some of which exhibit restriction fragment length polymorphism in different races of the pathogen. Western blot analysis of extracts from F. fulva with antibodies raised to purified reverse transcriptase (from human immunodeficiency virus-1) revealed immunoreactive proteins. Reverse transcriptase previously has been detected in a variety of organisms including yeast, insects, protozoa, and mammals, but to our knowledge, this is the first report of its occurrence in filamentous fungi.

Heterologous gene expression in Aspergillus niger: a glucoamylase-porcine pancreatic prophospholipase A2 fusion protein is secreted and processed to yield mature enzyme.

The cDNA gene encoding porcine pancreatic prophospholipase A2 (proPLA2) was cloned into an Aspergillus niger expression vector downstream of the glucoamylase (glaA) gene promoter region. When this construct was transformed into A. niger, no detectable PLA2 was produced. Evidence was obtained showing that the PLA2 gene was transcribed and that PLA2 is extremely susceptible to both intracellular and extracellular proteases of A. niger, thus indicating that translation products would be rapidly degraded. By fusing the proPLA2-encoding sequence to the entire glaA gene, secreted yields of PLA2 up to 10 micrograms/ml were obtained from a transformed protease-deficient strain of A. niger. PLA2 was secreted in young cultures as a fusion protein, but in older cultures, it was processed from the glucoamylase carrier protein. Secreted PLA2 was shown to be enzymatically active and to have the correct N-terminal amino acid (aa) sequence, although another form of processed PLA2 was also produced. This form included two aa of the proregion from PLA2. The potential for improving yields of secreted heterologous proteins from A. niger still further is discussed.

A pyruvate decarboxylase gene from Aspergillus parasiticus.

A gene encoding a putative pyruvate decarboxylase (EC 4.1.1.1) was isolated from a genomic library of the filamentous fungus Aspergillus parasiticus strain SU-1. The deduced amino acid sequence showed 37% homology to PDC1 from Saccharomyces cerevisiae. Although A. parasiticus has an obligate growth requirement for oxygen, it produced ethanol in shake flask cultures indicating a response to anoxic conditions mediated by pyruvate decarboxylase.

Nosocomial transmission of Saccharomyces cerevisiae in bone marrow transplant patients.

Saccharomyces cerevisiae is an unusual cause of clinical infection. We describe three bone marrow transplant patients on a haematology unit who developed possible invasive disease with the organism. Two patients died and both these patients appeared to have a related strain of S. cerevisiae. Screening for S. cerevisiae from throat and stool samples revealed four further patients who were carriers. Genotyping of the invasive and carriage strains demonstrated an indistinguishable strain from patients who had been on the unit at the same time, suggesting cross-infection.

Heterologous protein production by filamentous fungi.

There are clearly many facets to successful production of heterologous proteins from filamentous fungi. The objectives are to exploit the natural ability of some species to secrete high levels of protein. The heterologous target proteins produced in a fungal host must be acceptable to the public and be economic to produce, i.e. the targets must be authentic (in structure and activity) and be produced in high yield to necessary levels of purity. The appearance of heterologous products from fungi on the market is testament to some success but, equally, there are considerable limitations in our ability to produce desired yields of many target proteins. We endorse the view of van den Hondel, Punt and van Gorcom (1991) that for the commercial production of heterologous proteins from filamentous fungi more information is required on transcriptional control, introns, mRNA stability and processing, translational efficiency, protein secretion, glycosylation and proteolysis. In addition, there is scope for yield improvement based on a better understanding of the physiology of growth/product secretion coupled to appropriate bioreactor operation. The authenticity of product is an aspect which will assume increasing importance, particularly for therapeutic proteins. The level at which the structures and functional activity of heterologous proteins are assessed will ultimately be determined by legislation. The analytical methods currently available are not always sufficient, for example, to reveal folded structures, and most proteins are not amenable to analysis by two-dimensional NMR. The authenticity of target heterologous proteins will also need to be assessed in relation to the glycosylation level and pattern. This is not easily done and explains the paucity of detailed information published to date on glycosylation of fungal proteins. Novel engineered proteins are already being produced from filamentous fungi where expression is an aid to investigation of structure-function relationships. Commercial production of such engineered proteins will require approval subject to a range of stringently applied tests and analyses. This imposes an even greater need to be able to specify and control, in a rational manner, the structures of recombinant proteins. The research needs for realization of improved yields are equally important in assuring authenticity of product. It is encouraging that progress is being made on all fronts, primarily with Aspergillus spp. and T. reesei, but also with other species, such as N. crassa.

Pollen-stigma interactions in Brassica oleracea. I. Ultrastructure and physiology of the stigmatic papillar cells.

The osmotic potential (psi pi) of the stigmatic papillar cells of Brassica oleracea is -14.8 bars. In laboratory conditions each cell transpires water at rates within the range from 3 X 10(-5) to 5 X 10(-5) mm3h-1. A small increase in transpiration rate is detected following cross-(compatible) but not self-(incompatible)pollination. No significant changes in psi pi occur following pollinations of either compatibility. Electron microscopy reveals an active papillar cytoplasm apparently secreting proteins into the cell wall via small vesicles. The cuticle is discontinuous and freeze-fracture techniques indicate that channels transverse the cell wall, suggesting a possible pathway for the movement of protein molecules of high molecular weight from the cytoplasm to the stigma surface. Analysis of electron-microscopic autoradiographs of mature, self-incompatible papillae following pulse-chase experiments with L-[3H]leucine and treatment with cycloheximide shows that protein molecules secreted into the cell wall may return to the cytoplasm at a later stage. The significance of these results is discussed in terms of current models of the pollen-stigma interaction in Brassica.

Pollen-stigma interactions in Brassica oleracea. II. The fate of stigma surface proteins following pollination and their rôle in the self-incompatibility response.

Mature, self-incompatible stigmas exposed to cycloheximide for 2 h prior to pollination supported identical germination and growth of both cross and self pollen. Treatment of self-pollinated pistils with cycloheximide resulted in the germination of hitherto inactive pollen after some 2-4 h. Pollen germination and initial tube growth in an in vitro germination medium were not significantly affected by cycloheximide. A continuous synthesis of stigmatic proteins is therefore essential for the operation of the self-incompatibility (S.I.) system. However, light-microscope autoradiography of stigmas fed with L-[3H]leucine prior to pollination revealed no movement of stigmatic proteins into the pollen, independent of the compatibility of the pollen with respect to the stigma. Further, tunicamycin, when applied in the same way as cycloheximide, had no effect on the S.I. system. These results are discussed in terms of the proposed cycling of proteins in the papillar cell wall and the involvement of a stigmatic glycoprotein in the S.I. response.

Ethylene-induced microtubule reorientations: mediation by helical arrays.

Entire microtubule arrays, within outer cortical and epidermal cells of pea epicotyl and mung-bean hypocotyl, have been visualized by indirect immunofluorescence. In all cells the microtubule arrangement can be interpreted as being a single multistart helix of variable pitch. In control cells the predominant pattern is a tightly compressed helix with the microtubules consequently in a net transverse direction with respect to the cell axis. Occasionally some cells show an oblique helix and rare cells show a longitudinal array which may be interpreted as a steeply pitched helix. By contrast in ethylene treated tissue, many cells show net longitudinal and oblique arrays of microtubules and few show transverse arrays. Similar effects can be induced by high osmolality. It is suggested that the plant cortical cytoskeleton is an integral unit, capable of wholesale reorientation in response to environmental signals.

Pollen-pistil interaction in Brassica oleracea : Events prior to pollen germination.

Quantitative studies of the adhesion of pollen grains to the stigma in Brassica oleracea revealed that self-pollen is initially less firmly bound than cross-pollen. The pollen grain tryphine, believed to be important in the adhesion process, has been shown to differ in mobility following self- and cross-pollination when observed using fluorescent probes. The hydration of the pollen grains has been investigated in vitro by measuring the changes in shape, volume and fresh weight of the imbibing grains. Whilst little change in volume could be detected there was a considerable increase in fresh weight together with a change of shape. The significance of these events, which occur prior to pollen germination, is discussed in relation to their effect upon subsequent germination and expression of self-incompatibility.

Genetic interrelationship among species of the genus Zygosaccharomyces as revealed by small-subunit rRNA gene sequences.

The phylogenetic interrelationship of species of the genus Zygosaccharomyces was examined by 18S rRNA gene sequencing. Comparative analysis of the sequence data revealed the genus to consist of a number of distinct subdivisions. The most prevalent species associated with food spoilage, Z. bailii, Z. bisporus and Z. rouxii, along with Z. mellis were found to form one subdivision. Zygosaccharomyces cidri and Z. fermentati formed a distinct species pair, as did Z. microellipsoides and Z. mrakii. Zygosaccharomyces florentinus formed a separate line displaying no specific relationship with any of the other Zygosaccharomyces species examined. Comparison with nine published ascosporogenous yeast 18S rRNA gene sequences showed that Z. microellipsoides and Z. mrakii were genealogically very close to Torulaspora delbrueckii (both displaying 99.8% 18S rRNA sequence similarity), raising the possibility that these two Zygosaccharomyces species should be moved to the genus Torulaspora. The topologies of trees derived from complete 18S rRNA gene sequences and from individual domains within the gene were compared and the implications of using partial sequence data for inferring phylogenetic relationships discussed.

Phylogenetic heterogeneity of the genus Williopsis as revealed by 18S rRNA gene sequences.

A phylogenetic investigation of the ascomycetous yeast genus Williopsis was performed by using 18S rRNA gene sequence analysis. Comparative sequence analysis revealed the genus to be phylogenetically heterogeneous. The five varieties of Williopsis saturnus [var. mrakii, var. sargentensis, var. saturnus (type), var. suaveolens and var. subsufficiens] were found to have identical 18S rRNA gene sequences and formed a distinct group, quite separate from all other Williopsis and non-Williopsis species examined. Williopsis mucosa was found to be the closet phylogenetic relative to the Williopsis saturnus group, however a sequence divergence of approximately 2.3% suggests this species may belong to a separate genus. The recently described species Williopsis salicorniae was found to exhibit a relatively close association with Ogataea minuta (identical to Pichia minuta), the type species of the genus Ogataea. The remaining two members of the genus, Williopsis californica and Williopsis pratensis, were found to form distinct lineages, displaying no specific association with any other Williopsis or non-Williopsis species. Based on comparative analysis of 18S rRNA genes it is apparent that the genus Williopsis as presently constituted is not monophyletic, and that the five currently recognized species form separate sublines each potentially worthy of separate generic status. The genus Williopsis should be restricted to the type species Williopsis saturnus and its five varieties. Despite the five varieties of Williopsis saturnus being genealogically indistinguishable at the 18S rRNA gene level, sequence analysis of the Internal transcribed spacer (ITS) region revealed that the five varieties could be differentiated on both their ITS1 and their ITS2 sequences, providing further evidence of the value of ITS sequences for discrimination of yeasts at the subspecies level.

Phylogenetic relationships among members of the ascomycetous yeast genera Brettanomyces, Debaryomyces, Dekkera, and Kluyveromyces deduced by small-subunit rRNA gene sequences.

A molecular systematic investigation of members of the ascomycetous yeast genera Brettanomyces, Debaryomyces, Dekkera, and Kluyveromyces was performed by using 18S rRNA gene sequence analysis. Our comparative sequence analysis revealed that Brettanomyces anomalus and Brettanomyces bruxellensis were closely related to one another and also to their teleomorphs, Dekkera anomala and Dekkera bruxellensis, respectively. Together with Dekkera custersiana and Dekkera naardenensis, these four species formed a stable and distinct phylogenetic group. The three representative species of the genus Debaryomyces examined (viz., Debaryomyces castellii, Debaryomyces hansenii, and Debaryomyces udenii) were found to be genealogically highly related to each other and exhibited a specific phylogenetic affinity (level of sequence similarity, approximately 99.2%) with Candida guilliermondii (teleomorph, Pichia guilliermondii). Debaryomyces species and C. guilliermondii formed a distinct phylogenetic group, which displayed a significant association with a phylogenetically coherent cluster encompassing Lodderomyces elongisporus, Candida albicans, and four other Candida species. In contrast to the situation with the genera Brettanomyces and Debaryomyces, the genus Kluyveromyces displayed very marked phylogenetic heterogeneity. Kluyveromyces polysporus, the type species of the genus Kluyveromyces, and six other Kluyveromyces species (viz., Kluyveromyces africanus, Kluyveromyces delphensis, Kluyveromyces lodderae, Kluyveromyces thermotolerans, Kluyveromyces waltii, and Kluyveromyces yarrowii) were phylogenetically intermixed with species of the genera Zygosaccharomyces, Saccharomyces, and Torulaspora. In contrast, Kluyveromyces aestuarii, Kluyveromyces dobzhanskii, Kluyveromyces lactis, Kluyveromyces wickerhamii, and three Kluyveromyces marxianus varieties, along with their anamorph, Candida kefyr, formed a highly stable monophyletic group worthy of separate generic status. Kluyveromyces blattae and Kluyveromyces phaffii formed two distinct phylogenetic lines that did not exhibit particularly close affinity with each other or other ascomycetous yeast genera. Our phylogenetic findings are discussed in the context of the results of other genotypic and phenotypic studies.

Candida sorbosivorans sp. nov., a new member of the genus Candida Berkhout.

A yeast, strain NCYC 2938T, was isolated from contaminated industrial material. This material was involved in a cascade continuous process for oxidizing sorbitol (D-glucitol) to L-sorbose. The isolate is similar, although not identical, to Candida geochares and Candida magnoliae in its physiological characteristics. Sequence analysis of the 26S rDNA D1/D2 variable domain showed that it was similar to those of both Candida species, but differed sufficiently to be considered as a separate species. Both the physiological characteristics and the unique 26S rDNA D1/D2 sequence of NCYC 2938T are described here, and the yeast has been named Candida sorbosivorans sp. nov. The type strain is NCYC 2938T (= CBS 8768T).

Phylogenetic analysis of the psychrophobic yeast Arxiozyma telluris and the reinstatement of Candida pintolopesii (van Uden) Meyer et Yarrow and Candida slooffii van Uden et do Carmo Sousa.

A phylogenetic analysis was conducted upon ten strains of the psychrophobic yeast species Arxiozyma telluris using nuclear rDNA (18S and 26S) and mitochondrial cytochrome-c oxidase subunit II (COX2) gene sequences. Strains examined included those described originally as Candida slooffii, Torulopsis bovina (= Candida bovina) and Torulopsis pintolopesii (= Candida pintolopesii), which are all currently accepted as synonyms of Arxiozyma telluris. Comparative 18S rDNA sequence analysis showed that these strains formed a genealogically highly related group, which was phylogenetically distinct from any other ascomycetous species studied. The results showed that A. telluris, as currently described, appears to be composed of a complex of closely related but nevertheless separate taxa. rDNA and COX2 gene sequence data revealed that CBS 1787T, the type strain of C. pintolopesii, the currently recognized asexual form (anamorph) of A. telluris, along with strains CBS 2676 and CBS 2985 formed a distinct taxon that is phylogenetically separate from A. telluris. Similarly, the sequence data also showed that C. slooffii is a distinct taxon and support the reinstatement of this species. However, with regard to the relationship between the type strains of A. telluris (CBS 2685T) and C bovina (CBS 2760T), discrepancies were observed between the rDNA and COX2 sequence datasets, and these results are discussed in more detail.

The genetic relationship of Lodderomyces elongisporus to other ascomycete yeast species as revealed by small-subunit rRNA gene sequences.

The 18S rRNA gene sequence of the ascomycete yeast Lodderomyces elongisporus was determined by PCR-direct sequencing. The phylogenetic inter-relationship of Lodderomyces elongisporus and other ascomycete yeast species was examined by comparative sequence analysis. Lodderomyces elongisporus was found to be most closely related to Candida parapsilosis, C. tropicalis and C. albicans, exhibiting sequence similarity values of greater than 97.5%. The relationship between L. elongisporus and Candida parapsilosis in particular is discussed with regard to the possibility that L. elongisporus is the teleomorph (sexual form) of C. parapsilosis.

Use of an rRNA internal transcribed spacer region to distinguish phylogenetically closely related species of the genera Zygosaccharomyces and Torulaspora.

Analyses of the sequences of the small-subunit (18S) rRNA gene and two internal transcribed spacers (ITSs), ITS1 and ITS2, revealed that members of the yeast genera Torulaspora and Zygosaccharomyces are phylogenetically intermixed. Despite some minor differences in 18S rRNA-, ITS1-, and ITS2-derived trees, in general the patterns of the relationships inferred from the three chronometers were in good agreement. The ITS sequences of Torulaspora and Zygosaccharomyces species exhibited far greater interspecies differences than the 18S rRNA sequences and were better than 18S rRNA sequences for measuring close genealogical relationships. Despite the existence of interstrain ITS sequence variation in some species, it is possible to identify conserved regions in both ITSs that are useful in species differentiation.

Pollen-stigma interaction in Brassica oleracea: the role of stigmatic proteins in pollen grain adhesion.

The adhesion of pollen grains to the stigmas of Brassica oleracea was assayed after treatment of the stigmas wiuth protease and/or cycloheximide. Treatment with protease alone adversely affected pollen grain adhesion. However, the adhesive properties of the stigma recovered fully if the stigmas were not pollinated until 2 h after treatment. Immersion of the stigmas in cycloheximide after protease treatment prevented any recoveryt of the stigmas' adhesive properties. Cycloheximide treatment alone prevented pollen grain adhesion when pollination occurred later than 1--2 h after treatment but did not affect pollen grain adhesion if pollination occurred immediately after treatment. These results indicated not only that the surface-held proteins of the stigma are involved in pollen grain adhesion, but also that their turnover rate is rapid. Isoelectric focusing of extracts derived from stigmas after protease and cycloheximide treatment showed a marked decrease in staining intensity of 3 protein bands, one of which, a glycoprotein, is known to be present only when the self-incompativility system is fully functional. These observations suggest a specificity of adhesion between higher plant cells in the presence of the cell wall.

Expression of the Escherichia coli beta-glucuronidase gene in industrial and phytopathogenic filamentous fungi.

A chimaeric beta-glucuronidase (GUS) gene has been created by ligating the Aspergillus nidulans glyceraldehyde 3-phosphate dehydrogenase promoter to the coding sequence of the E. coli uidA gene. Co-transformation of this vector into A. nidulans, A. niger and the tomato pathogen Fulvia fulva (syn. Cladosporium fulvum (Cooke] resulted in the expression of beta-glucuronidase. GUS activity was detected by growth on agar media containing X-gluc and by enzyme assays of mycelial extracts. Expression of the gene in F. fulva transformants was also easily detectable during growth in plants and did not affect pathogenicity. These results form the basis for a versatile and sensitive reporter gene system for industrial and phytopathogenic filamentous fungi.

Pollen stigma interactions in Brassica oleracea.

Recent studies on the mechanism of self-incompatibility in Brassica indicate the location, nature and mode of action of the molecules involved. Characteristics of the pollen surface and the stigma surface are described in detail, together with new information pertaining to the recognition molecules located therein. A sequence of events is outlined leading from pollination, through adhesion, hydration, germination, and tube growth to acceptance and ultimate compatibility. The characteristics of rejection of incompatible grains are described for each stage of the pollen-stigma interaction. It is proposed that recognition of proteins from the coating of self-pollen by the molecules in the pellicle results in the formation of a biologically-active complex which inhibits water supply to the incompatible grain, and that all other manifestations of incompatibility are a consequence of this initial response.